【作者】 焦磊；

【导师】 吴开云；

【作者基本信息】 苏州大学 ， 人体解剖学， 2007， 硕士

【摘要】 研究目的：本课题通过研究NE对血管平滑肌细胞表型转化和增殖的影响及作用机制，为进一步探讨神经因素对VSMC表型转化和增殖作用打下基础。实验方法：取健康SD大鼠腹主动脉作原代培养，用第五代的细胞做实验。实验一：实验分为A：正常对照（control）组；B：NE（5×10-5.mol／L）作用组；C：NE+OX-LDL（5×10-5mol／L、50ug／ml）作用组；D：OX-LDL（50ug／ml）作用组。用0.5％血清饥饿同步化24小时后，各组换成无血清培养液并分别加入相应药物，作用24小时后收集细胞。用于免疫细胞化学检测的细胞，取出前24小时用5-Brdu(8×10^-4mol／l)标记，其它用于RT-PCR检测SM22amRNA和HRG-1mRNA的表达。实验二：实验分为A：72小时对照组；B：正常对照（control）组；C：NE（5×10-5mol／L）作用组；D：NE+OX-LDL（5×10-5mol／L、50ug／ml）作用组；E：OX-LDL（50ug／ml）作用组。用0.5％血清饥饿同步化24小时后，各组换成10％胎牛血清DMEM培养基培养72小时后，弃培养液换成无血清培养液（此时72小时对照组终止培养收集细胞），在C、D、E组中分别加入相应药物，作用48小时后收集细胞，其它用于RT-PCR检测SM22amRNA和HRG-1mRNA的表达。实验三：实验分为A：正常对照（control）组；B：a1-R^-(10^-4mol／l)+NE(5×10^-4mol／l)作用组；C：β1-R^-(10^-4mol／l)+NE(5×10^-4mol／l)作用组；D：NE(5×10^-4mol／l)作用组。用0.5％血清饥饿同步化24小时后，各组换成无血清培养液并分别加入相应药物，作用24小时后收集细胞。用于免疫细胞化学检测的细胞，取出前24小时用5-Brdu(8×10^-4mol／l)标记，其它用于其它用于RT-PCR检测SM22amRNA和HRG-1mRNA的表达。结果：1．NE对血管平滑肌增殖的作用NE和OX-LDL作用收缩型平滑肌细胞后，有下调HRG-1基因的表达，两者共作用下调更明显：Brdu标记也见其标记增殖细胞增多；而当NE和OX-LDL作用合成型平滑肌细胞时，NE能上调HRG-1基因，而OX-LDL则不能。这一结果表明NE对血管平滑肌的增殖有调控作用，而OX-LDL对平滑肌的增殖作用是不可逆的。而a1-R^-+NE和β1-R^-+NE组HRG-1mRNA的表达与NE作用组相比明显增多，Brdu标记增殖平滑肌细胞也大大减少，说明NE引起的血管平滑肌增殖是通过a1-R和β1-R起作用。2．NE对血管平滑肌表型转化的作用正常血管平滑肌都表达SM22a mRNA，NE和OX-LDL作用收缩型血管平滑肌后，SM22a mRNA表达都下调。而NE和OX-LDL作用合成型血管平滑肌时，NE对SM22a mRNA的表达有明显上调作用，OX-LDL则没有上调作用，这一点与对平滑肌的增殖作用是相吻合的；OX-LDL对合成型平滑肌的表型转化作用也是不可逆的。结论：1．NE可促收缩型血管平滑肌表型向合成型转化和增殖作用；对合成型血管平滑肌表型转化和增殖作用有回调作用，说明NE具有象神经调控样的可逆性调控作用。2．OX-LDL可促收缩型血管平滑肌表型向合成型转化和增殖作用：对合成型血管平滑肌表型转化和增殖没有看到象NE的回调作用，说明OX-LDL对血管是一种不可逆的作用。3．通过阻断a1-R和β1-R信号，完全可阻断NE对血管平滑肌表型转化和增殖作用，由此说明用NE对血管平滑肌表型转化和增殖作用是通过a1-R和β1-R来实现。4．对NE具有象神经调控样的可逆性调控作用，有必要作进一步更深入的研究。更多还原

【Abstract】 Objective: The influence of NE on the proliferation and transformation of phenotype ofvascular smooth muscle cells and its mechanism were studied to supply a experimentaldata and base for studying the effects of the sympathetic nerves on the proliferation andtransformation of phenotype of VSMC.Methods:The vascular smooth muscle cells from aorta of healthy SD rats were incubated incontaining 20% bovine serum culture medium for propagation of the experimental cellsand the fifth generation of cultured cells was used to this experiment.The first experiment: The experimental cells were divided into three experimentalgroup and one control group:A:normal control group; B: treat with NE （5×10-5mol/L）group; C: treat with NE （5×10-5mol/L） and OX-LDL （50ug/ml） group; D:treat withOX-LDL （50ug/ml） group. The experimental cells of each group were cultured incontaining 0.5% serum for 24 hours. Then the culture media were discarded and threeexperimental groups were replaced by NE, NE plus OX-LDL and OX-LDL respectively,and the control group was replaced by serum-free medium for continuous incubation for24h. As for the experimental cells used for immunocytochemical staining, thebromodeoxyuridine BrDU (8×10^-4mol/l) was added into the culture medium as sign dosagebefore 24h of accomplishing culture and others was used to detect the expression ofSM22aand HRG-1mRNA by RT-PCR.The second experiment: The experimental cells were divided into three experimentalgroup and two control group: A: 72h cotrol group; B:normal control group; C: treat withNE (5×10^-5 mol/L) group; D: treat with NE (5×10^-5 mol/L) and OX-LDL （50ug/ml） group; E: treat with OX-LDL （50ug/ml） group. The culture media of each group werereplaced into containing 20% serum media after cultured in containing 0.5% serum for 24hours. Then the culture media were discarded and three experimental groups were replacedby NE, NE plus OX-LDL and OX-LDL respectively, and the control group was replacedby serum-free medium for continuous incubation for 48h and the 72h control group wasstopped to culture.The experimental cells of each group was collected to detect theexpression of SM22a and HRG-1 mRNA by RT-PCR.The Third experiment: The experimental cells were divided into three experimentalgroup and one control group: A: normal control group; B: treat with a1-R^- (10^-4mol/l)and NE (5×10^-4mol/l) group; C: treat withβ1-R^- (10^-4mol/l) and NE (5×10^-4mol/l)group; D: treat with NE (5×10^-4mol/l) group. The experimental cells of each group werecultured in containing 0.5% serum for 24 hours. Then the culture media were discardedand three experimental groups were replaced by a1-R^- and NE,β1-R^- and NE and NErespectively, and the control group was replaced by serum-free medium for continuousincubation for 24h. As for the experimental cells used for immunocytochemical staining,the bromodeoxyuridine BrDU (8×10^-4mol/l) was added into the culture medium as signdosage before 24h of accomplishing culture and others was used to detect the expression ofSM22aand HRG-1mRNA by RT-PCR.Results:1.Effects of NE on the proliferation of vascular smooth muscleThe mRNA expression of HRG-1 was decreased after VSMC of contractilephenotype treated by NE and OX-LDL and a lot of proliferous cells labeled by Brdu can beseen. When VSMC of synthesize type treated with NE and OX-LD respectively, it can beseen that the mRNA expression of HRG-1 was up regulative by NE but not by OX-LDL.The results demonstrate that NE can regulate the proliferation of VSMC, however, effect ofOX-LDL on VSMC aren’t reversed. The mRNA expression of HRG-1 in treat with a1-R^-andβ1-R^- groups is more than in treat with NE group, and the number of proliferous cellslabeled by Brdu is getting little. This results show that the effect of NE on proliferation ofVSMC come true by means of a1-R andβ1-R function. 2.Effects of NE on tansformation of phenotype of VSMCAll normal VSMC show the expression of SM22amRNA, but the SM22amRNAexpression had apparent decreased after VSMC of contractile type treated by NE andOX-LDL. However, when VSMC of synthesize type treated with NE and OX-LDLrespectively, the mRNA expression of SM22a was up regulative by NE but not byOX-LDL. The results demonstrate that NE can regulate transformation of phenotype ofVSMC, however, effect of OX-LDL on transformation of phenotype of VSMC isn’treversed.Conclusions;1. NE can promote the proliferation and transformation of phenotype of VSMC fromcontractile to synthesize type. It also can regulate the proliferation and phenotype ofsynthesize type VSMC. This demonstrates NE is reversible regulating VSMC as vascularnerve function.2. OX-LDL can promote proliferation and transformation of phenotype of contractile typeVSMC, but it was not seen as the effect of NE on the proliferation and transformation ofphenotype of VSMC, when VSMC treat with OX-LDL. This showed that effect ofOX-LDL on VSMC isn’t reversed.3. It is completely interrupted the effect of NE on proliferation and transformation ofphenotype of VSMC after using a1-R and 1β-R, which shows that effects of NE onproliferation and transformation of phenotype of VSMC come true by means ofa1-R andβ1-R.4. It is necessary to make furthur research of the effect of NE on VSMC as vascular nervefunction.更多还原